The transcriptional machinery that regulates activated transcription in human cells involves basal factors, activators, coactivators, repressors and chromatin remodeling complexes, all acting of DMA within the context of a variegated chromatin structure. We have shown that the modular protein, HMGB1, can exhibit properties of both a coactivator (on regulatory factors) and a repressor (on TBP), showing context-dependent transcriptional regulation. Although HMGB1 was shown to enhance binding of steroid hormone receptors generally, we 1) showed that HMGB1 greatly enhances the binding affinity of estrogen receptor alpha, to estrogen response element (ERE) half-sites (HERE), to which ER binds only weakly without HMGB1 and 2) characterized these complex. We will continue the studies with three specific aims. We shall examine a number of additional HEREs, including tandem HEREs that are in "inverted, everted or direct" repeats and determine the effect of HMGB1 on the binding affinity for ERalpha and ERbeta {ER). We shall prepare HEREs in translationally and rotationally positioned nucleosomes to determine the ER binding affinity in this context at two different translational positions and how this compares to ER binding to the naked DMA sites. The effect of HMGB1 on the nucleosomes stability and the effect on ER binding at these sites will also be of importance and will be determined. We shall then determine if the increased in vitro ER binding affinity in the presence of HMGB1 translates into effects on transcription by carrying out transient transfections and monitoring the transcriptional CAT reporter assays using a variety of EREs and HEREs, in the presence and absence of overexpressed HMGB1. These findings will help to determine the role of HEREs in the genome and the effect that HEREs and HMGB1 may exert in the regulatory scheme of estrogen-dependent gene regulation. [unreadable] [unreadable]